Modeling Cataract Surgery in Mice
Summary
This method models cataract surgery in vivo by removing lens fiber cells from adult mice and leaving behind the capsular bag with attached lens epithelial cells (LECs). The injury response is then assessed at various times post-surgery using molecular and morphological criteria.
Abstract
Cataract surgery (CS) is an effective treatment for cataracts, a major cause of visual disability worldwide. However, CS leads to ocular inflammation, and in the long term, it can result in posterior capsular opacification (PCO) and/or lens dislocation driven by the post-surgical overgrowth of lens epithelial cells (LECs) and their conversion to myofibroblasts and/or aberrant fiber cells. However, the molecular mechanisms by which CS results in inflammation and PCO are still obscure because most in vitro models do not recapitulate the wound healing response of LECs seen in vivo, while traditional animal models of cataract surgery, such as rabbits, do not allow the genetic manipulation of gene expression to test mechanisms. Recently, our laboratory and others have successfully used genetically modified mice to study the molecular mechanisms that drive the induction of proinflammatory signaling and LEC epithelial to mesenchymal transition, leading to new insight into PCO pathogenesis. Here, we report the established protocol for modeling cataract surgery in mice, which allows for robust transcriptional profiling of the response of LECs to lens fiber cell removal via RNAseq, the evaluation of protein expression by semi-quantitative immunofluorescence, and the use of modern mouse genetics tools to test the function of genes that are hypothesized to participate in the pathogenesis of acute sequelae like inflammation as well as the later conversion of LECs to myofibroblasts and/or aberrant lens fiber cells.
Introduction
The lens is a highly organized, transparent tissue that refracts light to produce a clearly focused image onto the retina1,2,3. This specialized organ is surrounded by an uninterrupted basement membrane (the capsule), which isolates the lens from other parts of the eye. The inner anterior surface of the capsule anchors a monolayer of lens epithelial cells (LECs), which then differentiate at the lens equator into lens fiber cells, which comprise the vast majority of the lens3. A cataract occurs when the lens loses its transparency due to factors like aging, genetic mutation, UV radiation, oxidative stress, and ocular trauma4. Cataract is a leading cause of blindness worldwide, particularly in countries with poor medical care5. However, this condition is now readily treated by surgical removal of opaque lens cells by phacoemulsification in which the central lens capsule and attached lens epithelium are removed, followed by the use of a vibrating probe to break the cellular mass of the lens into smaller fragments that can be suctioned out; leaving behind a capsular bag with some attached equatorial LECs1. Vision is then most commonly restored by post-surgical implantation of an artificial intraocular lens (IOL).
While cataract surgery (CS) is a highly effective and minimally invasive treatment for cataracts, a patient's post-surgical recovery can be acutely hampered by the development of ocular inflammation5,6,8. This inflammation can cause post-surgical pain, retinal edema leading to retinal detachment, as well as exacerbation of other inflammatory and fibrotic conditions like uveitis and glaucoma4,7,8,9. Ocular inflammation post-CS (PCS) is generally treated with anti-inflammatory eye drops which are plagued by poor patient compliance or dropless cataract surgery that can lead to an increased prevalence of floaters8,10,11. In the long term, the outcomes of CS can be compromised by the development of posterior capsular opacification (PCO)12. PCO occurs when residual LECs left behind after surgery undergo a wound healing response, proliferate, and migrate onto the posterior lens capsule at months to years PCS contributing to secondary visual obstruction13,14.
Understanding the molecular mechanisms by which CS results in such acute and chronic responses represents a major challenge in the field, as much of the literature investigating PCO relies on the induction of LEC conversion to myofibroblasts in culture via treatment with active transforming growth factor beta (TGFβ)12,15. Human capsular bag models created from cadaver lenses cultured in vitro better reflect the biology of the lens PCS as LECs are cultured on their native basement membrane but are difficult to manipulate mechanistically, do not recapitulate the intraocular environment, and are inherently variable due to lens to lens (or donor to donor) variability16,17. Rabbit1,18 and non-human primate19,20 in vivo models of cataract surgery overcome some of these issues but still are not easy to manipulate for mechanistic studies. Notably, a prior study of mammalian lens regeneration found significant lens regeneration within four weeks after lens fiber cells were removed from mice, leaving the lens epithelial cells and capsule behind, while no lens regrowth occurred when the entire lens was removed21,22. We subsequently streamlined this procedure and optimized it for the study of the acute response of LECs to lens fiber cell removal and their subsequent conversion to a mixed population of cells with myofibroblast and fiber cell properties.
Using this mouse model of cataract surgery, we have shown that lens epithelial cells drastically remodel their transcriptome by 6 hours PCS to produce numerous proinflammatory cytokines23, while they begin expressing fibrotic markers as early as 24 h PCS12,23, prior to the onset of canonical TGFβ signaling. Mechanistic experiments in knockout mice using this model revealed that cellular fibronectin expression by LECs is required for a sustained fibrotic response PCS, likely due to both its role in the assembly of the fibrotic ECM and cell signaling12. Other studies showed that αVβ8-integrin is required for the transition of LECs to myofibroblasts due to its function in TGFβ activation, and the potential of this approach to identify anti-PCO therapeutic leads was confirmed as a αVβ8-integrin antagonist also blocked LEC EMT15.
Here, we provide a detailed protocol describing how to remove the lens fiber cells from living mice (Figure 1and Figure 4) while leaving behind the lens capsule and LECs to model the LEC response to cataract surgery.
Protocol
Representative Results
Discussion
This surgical technique requires advanced mouse handling and micro-surgical skills that take practice to develop. The most difficult to master is the placement of fine sutures in the cornea to close the wound. Suppose the experimenter is a total novice to suturing. In that case, it is recommended to first practice using string and cloth, then move to practice using large-diameter sutures on small fruits to master the needed hand movements to make surgical square knots. Then, the experimenter must practice making incision…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the National Eye Institute (EY015279 and EY028597) and Delaware INBRE (P20 GM103446).
Materials
| 0.5% Erythryomycin opthalmic ointment USP | Baush Lomb | 24208-910-55 | |
| 1% Atropine sulfate ophthalmic solution | Amneal | 60219-1748-2 | |
| 1% Tropicamide opthalmic solution USP | Akorn | 17478-102-12 | |
| 10-0 Nylon suture | Ethicon | 7707G | |
| 2.5% Phenylephrine hydrochloride ophtahlmic solution USP | Akorn | 17478-200-12 | |
| 26 G 1/2 Needles – straight | BD PrecisionGlide | 5111 | |
| 27 G 45° bent dispensing tip 1" | Harfington | ||
| Balanced saline solution | Phoenix | 57319-555-06 | |
| Bupranorphine (0.1 mg/kg) | APP Pharmaceticals | 401730D | |
| Chlorhexidine solution | |||
| Needle holder 2-110 | Duckworth & Kent | 2-110-3E | |
| Noyes scissors, straight | Fine Science Tools | 12060-02 | |
| SMZ800 Nikon model microscope | Nikon | ||
| Sterile disposable scalpel No.11 | Feather | 2975#11 | |
| Tweezers #5 Dumont | Electron Microscopy Sciences | 72700-D | |
| Xylazine/Ketamine/Acepromazine (35 mg/kg, 80 mg/kg, 0.5 mg/kg) solution | APP Pharmaceticals | 401730D |
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